Post-mortem Diagnosis of Heme Oxygenase-1 Deficiency by Whole Exome Sequencing in an Iranian Child

Heme oxygenase-1 (HO-1) is an inducible enzyme involved in the catalysis of heme conversion into biliverdin. We describe a patient with a novel stop-gain mutation in the HMOX1 coding sequence resulting in HO-1 deficiency. A 17-month-old female with fever, tachypnea, and signs of respiratory distress was referred to our center. Four admissions ensued during the eight months follow up. At the first admission, she had massive pericardial effusion without any laboratory findings for tuberculosis, viral infectionsor malignancies.An abdominal ultrasound examination confirmed hepatomegaly.Laboratory findings showed leukocytosis, thrombocytosis, hemolytic anemia, elevated inflammatory markers, increased levels of the hepatic transferase, triglycerides and ferritin as well as decreased level of fibrinogen. Other laboratory investigations were negative blood cultures, normal bone marrow aspiration, and normal serology viral infections. Immunodeficiency and auto-inflammatory syndromes were ruled out. Hepatic biopsy showed iron deposits. The patient was initiated on corticosteroids; however, her clinical condition was progressively deteriorated, and she died of recurrent fever, bleeding, heart failure, and ascites. Post-mortem whole exome sequencing revealed a homozygous mutation (exon3: c.A610T, p.K204X) on the HMOX1 gene. The parents were found to be heterozygous for that mutation. The laboratory findings and clinical features of our patient were somehow similar to that of HO-1 deficient cases reported previously, as well as Hmox1 knocked out mice. We speculate that the clinical manifestations of HO-1 deficient patients can be partially dependent on the type of mutation they inherit.

HO-2 have been assigned to chromosome 22q12 and 16p13.3, respectively (3). The HMOX1 gene contains five exons and encodes a 288 amino acid protein. These enzymes catalyze the oxidative conversion of heme into biliverdin, carbon monoxide, and iron (3). The constitutive expression of HMOX2 has been demonstrated in different tissues; however,HMOX1 has been shown to be induced in response to its substrates, including heme and heme-derived metabolites (4,5).
In spite of its action in heme conversion, it has also been demonstrated that HO-1 has an important antioxidant role (2,6). In this regard, different studies have suggested that HO-1 plays a protective role in various inflammatory conditions (2,6,7).
One of the well-known mechanisms by which HO-1 exerts antioxidant effects is the production of biliverdin. Biliverdin is then reduced and metabolized into bilirubin by another enzyme, which is a crucial endogenous antioxidant and protects tissues from damage induced by a variety of oxidative stressors (8,9).
As the heme degradation is mainly occurring in the liver and spleen, the highest expression of HMOX1has also been detected in these tissues (5,10,11). Several studies have shown that a high level of HO-1 is produced particularly in hepatic and splenic tissue-resident macrophages in response to inflammatory mediators(5, 10-12). These macrophages have an anti-inflammatory phenotype and are the primarycells that respond to oxidative injury. Therefore, the impaired function of HO-1 or its deficiency can hamper the function of these cells, which aggravates tissue injury through an inflammatory cascade. Furthermore, these findings demonstrated that any defect in the iron recycling system could result in health-threatening issues (13,14). A remarkable consistency was observed between the clinical features of HO-1 deficient patients, and those findings obtained from Hmox1knocked out mice.
The first patient with HO-1 deficiency (OMIM#614034) has been reported by Yachie et al. (15). Serum iron deficiency and iron overload were initial findings for HO-1 deficiency.
Moreover, the findings revealed that patients with HO-1 deficiency suffered severely from high fever, massive endothelial injury, abnormal coagulation system, extensive hemolysis as well as liver and kidney failure (15).
Here, we report a new case of HO-1 deficiency who carried a novel stop-gain mutation in the HMOX1coding sequence. We also performed a literature review to compare the laboratory findings, and clinical features of our patient with those patients that have been reported previously, and Hmox1-targeted mice.

Case presentation
The patient was a 17-month-old girl with 9200 g weight, 71 cm height, and 46 cm head circumference who was referred to Children's Medical Center in Tehran, Iran. She was born to consanguineous Iranian parents with a birth weight of 3200 g, length of 50 cm, and head circumference of 45 cm.She was the third child of a 28-year old mother whose delivery was performed by cesarean section at week 38. The first child of this family was a girl who was found to be completely healthy, The patient was admitted again three months after the second admission since fever has recurred.
No histological and serological evidence was observed for arthritis. Echocardiographs and chest X-ray findings were essentially normal. Bone marrow aspiration was also performed, and no abnormality was seen. Similar to previous admission, there were increased levels of AST,  Table 1.
After five days, she was admitted once again because of a high fever. The fever and other symptoms resolved by corticosteroids and cotrimoxazole. Unfortunately, after a short remission, the clinical condition of the patient was progressively deteriorated, and she has died of recurrent fever, hemorrhage, heart failure, and ascites.
The clinical and laboratory findings (fever, hepatomegaly, iron deposits in liver cells, elevated ferritin level as well as elevated levels of liver enzymes (Table 1) have led us to suspect hemophagocyticlymphohistiocytosis (HLH) and HO-1deficiency.In this regard, to identify the genetic cause(s) responsible for this phenotype, we performed post-mortem whole exome sequencing followed by mutation confirmation by direct Sanger sequencing on the blood sample taken from the  jcvi.org/ downloads. php).

Discussion
Here, we describe a new case of HO-1 deficiency. Our patient presented with fever, leukocytosis, thrombocytosis, anemia, and increased levels of AST, ALT, ALP, TG, ferritin, ESR, CRP, and CpK (Table 1) To the best of our knowledge, there are only two previous human case reports on the HO-1  In conclusion, we report a new case of HO-1 deficiency presenting with HLH like presentation.
The phenotype of our patient was compatible with those two patients reported previously. In contrast to previous HO-1 deficient cases who exhibited asplenia, our patient has normal spleen mass. This case shows that there may be a partial difference between the pathophysiological findings of the cases with the same disease, which might result from the type of mutation they inherit.

Consent
Written informed consent was obtained from the parents of the patient for the publication of this case report.

Conflict of interest
The authors declare that they have no Competing interests.